Method for detection of target nucleic acid

ABSTRACT

An object of the disclosure of the present specification is to provide a method for detection of a target nucleic acid which allows construction of an effective detection system of a target nucleic acid. For this purpose, in the disclosure of the present specification, a first primer comprising an identification sequence complementary to a target sequence in a target nucleic acid and a tag addition sequence, and a second primer having a label are prepared. The first primer and the second primer are used for the target nucleic acid in a sample to amplify a chimeric DNA having a tag sequence and the label. The chimeric DNA is hybridized with a detection probe on a solid phase to obtain signal intensity information based on the label, and the target nucleic acid is detected based on the signal intensity information.

This application is a national phase entry under 35 U.S.C. §371 of PCTPatent Application No. PCT/JP2010/068964, filed on Oct. 26, 2010, whichclaims priority under 35 U.S.C. §119 to Japanese Patent Application No.2009-249122, filed Oct. 29, 2009, both of which are incorporated byreference. Also, the Sequence Listing filed electronically herewith ishereby incorporated by reference (File name:1027-0022_Seq_List_(—)2012-03-19; File size: 18 KB; Date recorded: Mar.19, 2012).

TECHNICAL FIELD

The present application claims priority to Japanese Patent ApplicationNo. 2009-249122 filed on Oct. 29, 2009, which is incorporated herein byreference in its entirety. The present invention relates to a technologyfor detecting target nucleic acids.

BACKGROUND ART

It has been conventionally proposed to exhaustively detect or quantitatenucleic acid sequences in order to carry out genetic analyses ofindividual organisms and to test an infection of biological samples withviruses or bacteria. For example, microarrays (hereinafter merelyreferred to as “arrays”) are used for detecting an expression level ofnucleic acid sequences to be detected (target nucleic acids) in samples(e.g., see Non-patent documents 1 to 4) Arrays are carriers on whichmultiple nucleic acid fragments (detection probes) having known basesequences are independently fixed. As shown in FIG. 8, in conventionalarray methods, a forward primer (F primer) and a reverse primer (Rprimer) designed so as to flank the target sequence are used to amplifya DNA fragment (target nucleic acid) containing the target sequence. Theamplified nucleic acid is then separated to a single strand. The targetnucleic acid then binds on the carrier by hybridization of the targetsequence with a portion complementary to a partial sequencecharacteristic to the target nucleic acid (target sequence). Thehybridized target nucleic acid is detected by any suitable method todetermine a presence or absence of the nucleic acid in the sample.Arrays specific for detection of single nucleotide polymorphisms (SNPs)have been developed (e.g., see Patent documents 1 and 2). By thismethod, a type of SNPs of the target nucleic acid in the sample can bedetected by using DNA computer technology.

CITATION LIST Patent Literature

-   Patent document 1: Japanese Patent Application Laid-open No.    2006-211982-   Patent document 2: Japanese Patent Application Laid-open No.    2006-101844

Non Patent Literature

-   Non-patent document 1: Baio Jikken de Shippai Shinai!Kenshutsu to    Teiryo no Kotsu (Successful Biotechnological Experiments: Tips for    Detection and Quantification), Supplementary volume of Jikken Igaku    (Medical Experiments), Yodosha, Chapter 3, 10. Maikuroarei no Kotsu    (Tips for Microarrays)-   Non-patent document 2: Bioview, No. 45, pp 14-18, 2004, Takara-Bio-   Non-patent document 3: Biotechnology series: DNA chip application    technology, CMC Publishing, Chapter 5, Practice an application of    DNA microarrays-   Non-patent document 4: Ministry of Health, Labour and Welfare    Grant-in-Aid for Scientific Research, Research project on securement    of food safety and reliability (Annual Report of Ministry of Health,    Labour and Welfare, 2006)

SUMMARY

In the methods disclosed in Non-patent documents 1 to 4, the detectionprobe fixed on the array is hybridized with the target sequence, whichhybridization requires a prolonged period of time. In addition, thedetection probe may bind non-specifically to other nucleic acidsequences having similarity (homology) with the target sequence. Namely,upon detection of multiple target sequences in the sample, the presenceor absence thereof may not be accurately detected.

Regarding the non-specific binding problems, Non-patent document 2discloses that homology of the detection probe can be minimized byreducing a length thereof. However, the reduction in the length of thedetection probe may decrease an intensity of signal of a label upondetection. Non-patent document 4 discloses that non-specific binding maybe decreased by increasing hybridization temperature. However, when theproblem is not solved by these methods, the sequence of the detectionprobe needs to be re-designed and the array needs to be re-prepared.Thus, users of arrays need to consider an influence of homology, makingprocess steps for obtaining an appropriate detection system for thetarget nucleic acid significantly intricate.

On the other hand, the methods disclosed in Patent documents 1 and 2 arespecific for detection of SNPs, which allow accurate detection of SNPs.However, seven different probes are required for the detection of oneSNP and procedures are further intricate. In addition, to design probesis troublesome because it is required for the user to ligateamplification byproducts of the target nucleic acid before hybridizationof the target nucleic acid to the array.

As described above, a lot of effort is required in conventional methodsfor constituting the detection system of intended target nucleic acids.It has been also difficult to accurately detect the target nucleic acidin a short time. Accordingly, an object of the disclosure of the presentspecification is to provide a method for detection of the target nucleicacid allowing effective construction of the detection system of thetarget nucleic acid.

The present inventors have studied in order to effectively construct thedetection system on various methods which allow effective hybridizationof the detection probe fixed on the carrier with the target nucleic acidwhile maintaining selectivity. As a result, they have reached to aconclusion that it is difficult to effectively construct the detectionsystem based on a hybridization reaction due to sequence specificity ofthe target nucleic acid on a solid carrier. They have also found thatconsideration on hybridization conditions may be omitted andnon-specific binding may be excluded and high selectivity can beachieved by using multiple sets of detection probes and tag sequenceswhich have been designed so as to be able to specifically hybridize andattaching the tag sequences to the target nucleic acid. In addition,without requiring ligation of such a chimeric target nucleic acid usinga probe specific to the target sequence, non-specific binding betweenthe labeled target nucleic acid and the detection probe can be reducedby amplifying the labeled target nucleic acid using primers specificallyhybridizable to a partial sequence having low homology, i.e. a sequencecharacteristic to the target nucleic acid. The following method isprovided based on these findings.

The disclosure of the present specification relates to a method fordetection of the target nucleic acid in the sample. The present methodfor detection comprises steps of preparing a solid phase comprisingdetection probes respectively having certain different base sequences,carrying out PCR on the sample to obtain chimeric DNAs each having alabel and a tag sequence complementary to each of the detection probeshaving been correlated to the target nucleic acid, bringing the chimericDNAs into contact with the detection probes such that the chimeric DNAsand the detection probes can hybridize through the tag sequences,obtaining signal intensity information based on the label on the solidphase, and detecting the target nucleic acid based on the signalintensity information.

The step of PCR comprises preparing a first primer having anidentification sequence complementary to the target sequence in thetarget nucleic acid and a tag addition sequence complementary to the tagsequence, and a second primer having a partial sequence identical to apartial sequence adjacent to the target sequence and the label, andcarrying out PCR on the sample using the first primer and the secondprimer to synthesize the chimeric DNA having the target sequence, thetag sequence and the label.

In the step of PCR, two or more first primers and one second primercommon to the two or more target nucleic acids may be used for two ormore target nucleic acids.

The step of PCR may be the step of amplifying the chimeric DNAs byasymmetric PCR.

The target nucleic acid can be detected by using the array comprisingthe detection probe hybridizable to the tag sequence having beencorrelated to the target nucleic acid.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view of an example of the method for detection ofthe present invention;

FIG. 2 is a view depicting relationship between the solid phase carrier,and the detection probes and the chimeric DNA on the solid phase carrieraccording to the present invention;

FIG. 3 is a view showing the step of amplification of the labeled targetnucleic acid according to the preset invention;

FIG. 4 is a flow chart for preparation of the array and the target;

FIG. 5 is a table showing base sequences of detection probes;

FIG. 6 is a view showing detection results obtained in examples of thepresent invention;

FIG. 7 is a view showing detection results obtained in examples of theconventional method; and

FIG. 8 is a view showing an exemplary conventional detection method oftarget nucleic acids.

DESCRIPTION OF EMBODIMENTS

The present invention relates to the array for detection of the targetsequence in the target nucleic acid which is to be detected. Accordingto the method for detection of the target nucleic acid of the presentinvention, a procedure can be avoided for constructing the detectionsystem by designing the detection probes having different unique basesequences respectively for all target nucleic acids and fixing them onthe solid phase carrier. By carrying out PCR on the sample so as toobtain the chimeric DNA having a detection sequence complementary to thedetection probe having been correlated to the target nucleic acid andthe label, the target nucleic acid can be identified and the chimericDNA which has the detection sequence having been correlated to thedetection probe, is specific to the target nucleic acid and is labeledcan be obtained by PCR for preparation of DNA for hybridization. Byhybridizing the chimeric DNA and the detection probe via the detectionsequence, the chimeric DNA hybridizes to the detection probe based onthe detection probe and the detection sequence which have beencorrelated to each other, effectively suppressing or avoidingnon-specific binding upon hybridization.

According to the disclosure of the present specification, the firstprimer having the identification sequence complementary to the targetsequence in the target nucleic acid and the tag addition sequence andthe second primer having the partial sequence adjacent to the targetsequence and the label are used when PCR is carried out, therebysuppressing or avoiding complicated design of probes or primers. The setof primers allows easy preparation of the chimeric DNAs forhybridization with the detection probes which directly identify thetarget sequences and are specific to the target nucleic acids.

This method ensures detection of each of multiple target nucleic acidsand can be used for detection of single nucleotide polymorphisms (SNPs)or modified sites in genetically modified nucleic acids or for detectionof expression genes such as RNAs. Namely, SNPs, modified sites,expression genes, polymorphisms or mutations in the target nucleic acidscan be detected by obtaining chimeric DNAs based on the same concept asthe detection of the mutation to be detected.

FIG. 4 shows an outline of the procedures of the preparation of thearray and the target for detection of the target nucleic acid. FIG. 4also shows the flow chart for the method disclosed in the presentspecification as well as the conventional detection method. The stepsshown with the solid line are the steps common for the method disclosedin the present specification and the conventional method, and the stepsshown with the dashed line are the ones necessary only for theconventional method. In the conventional method, multiple steps arecarried out for preparation of arrays for respective target nucleicacids. Information on the sequence of the target nucleic acid to bedetected is first obtained and the detection probe is designed accordingto the sequence information. The detection probe is then synthesizedaccording to the design and prepared in a spot solution for the array inorder to fix the probe on the array. Meanwhile the target nucleic acidto be detected (a target such as RNA or DNA) is extracted and purifiedfrom the sample. Primers for amplifying the target nucleic acid aredesigned and synthesized with a labeling. The target nucleic acid isthen amplified with the synthesized primers. The amplified targetnucleic acid is then hybridized with the detection probe on the arrayprepared (hybridization). The occurrence of hybridization is examinedaccording to detection of signal of the label on the array and theobtained signal of the label is converted to the numeral value. When theobtained results are not the ones expected such as abnormal or absenceof fluorescence signal etc., the array has to be designed again or thesample has to be prepared again, as shown with the solid and dashedlines in FIG. 4.

The conventional methods disclosed in Non-patent documents 1 to 4require many reviews on hybridization conditions, primers, and evensequences of the detection probes on the array. It takes a prolongedperiod of time to re-design and synthesize oligo DNAs for the detectionprobes and query probe sequences. According to the present method, thedetection probes and query probes may be merely selected from 100different sequences (see Sequence Listing). The methods disclosed inPatent documents 5 and 6 require seven different primers and probes fordetection of one target sequence. However, the present inventionrequires only two kinds of primers for detection of one target sequence.

On the other hand, the method disclosed in the present specificationmerely requires preparation of the array comprising multiple detectionprobes respectively having the unique detection sequence preliminarilydetermined regardless of the target nucleic acid. As the array can beapplied regardless of the target nucleic acid, design, synthesis andfixation of probes for respective target nucleic acids and review ofhybridization conditions may all be avoided, unlike the conventional amethod. The detection system may be constructed according to the methoddisclosed in the present specification by mainly considering only thedesign of the primers upon preparation of the target.

According to the method disclosed in the present specification, thedetection probes can be prepared for which hybridization conditions areoptimized, thus the target nucleic acid can be accurately detected in ashort time.

As used herein, the “nucleic acid” includes all DNAs and RNAs includingcDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA and syntheticRNA as well as artificial synthetic nucleic acids such as peptidenucleic acid, morpholino-nucleic acid, methylphosphonate-nucleic acidand S-oligo nucleic acid. The nucleic acid may be single-stranded ordouble-stranded. As used herein, the “target nucleic acid” is anynucleic acid having any sequence. Typically, the target nucleic acidincludes nucleic aids which may have base sequences geneticallyindicative for constitution or disease incidence, disease diagnosis,disease prognosis, drug or treatment selection of specific diseases suchas genetic diseases or cancer in human or non-human animals. Thegenetically indicative base sequences include polymorphisms such as SNPsand inherent or acquired mutations. The target nucleic acid alsoincludes nucleic acids derived from microorganisms such as pathogens andviruses.

The target nucleic acid may be the sample described below or a nucleicacid fraction thereof and is preferably an amplified product in whichall of the multiple target nucleic acids have been amplified bypreferably amplification reaction with PCR, more preferably multiplexPCR.

As used herein, the “sample” refers to the sample which may contain thetarget nucleic acid. The sample may be any sample containing a nucleicacid including cells, tissues, blood, urine, saliva and the like. Aperson skilled in the art may appropriately obtain a fraction containingthe nucleic acid from such various samples according to the conventionalart.

As used herein, the “target sequence” refers to a sequence formed by oneor more bases characteristic to the target nucleic acid to be detected.The target sequence may be a partial sequence having low homology amongthe target nucleic acids or a sequence having low complementarity orhomology to other nucleic acids which may be contained in the sample.The target sequence may be a sequence characteristic to the targetnucleic acid. The target sequence may have a sequence artificiallymodified.

Representative and non-limiting specific examples of the disclosure ofthe specification are described herein after with referring to thedrawings. The detailed description merely intends to illustrate thedetails to a person skilled in the art for carrying out the preferredexamples of the disclosure of the present specification, while it doesnot intend to limit the scope of the disclosure of the presentspecification. Additional features and disclosures hereinafter may beused separately or in conjunction with other features or inventions inorder to provide a further improved method for detection of the targetnucleic acid and the like.

Combinations of the features and steps disclosed hereinafter in thedetailed description are not requisite for carrying out the disclosureof the present specification in its broadest meaning, but areparticularly described merely for illustrating representative specificexamples of the disclosure of the present specification. Variousfeatures of the above- and below-described representative specificexamples as well as various features of those described in independentand dependent claims are not the ones which have to be combined as thespecific examples or in the same order as described herein in order toprovide additional and useful modes of the disclosure of the presentspecification.

All features described in the present specification and/or claims intendto be disclosed, individually and independently each other, aslimitations for specific items described in the disclosure and claims atthe time of filing the present application, separately from a structureof the features described in examples and/or claims. Descriptions on allnumerical ranges and groups or sets intend to disclose intermediateaspects thereof as limitations for specific items described in thedisclosure and claim at the time of filing the present application.

FIG. 1 is a schematic view showing a principle of the method fordetection of the present invention. FIG. 2 shows an example of the solidphase 100 used for the present invention and FIG. 3 shows details forthe step of amplification in FIG. 1. FIGS. 1 and 3 show an example forthe method for detection and the primer for detecting one target nucleicacid contained in the sample. In the descriptions hereinafter, a basesequence designated with a number and (-) means a complementary basesequence of a base sequence designated with the same number.

[Method for Detection of the Target Sequence in the Target Nucleic Acid]

The method for detection disclosed in the present specificationcomprises steps of preparing the solid phase comprising multipledetection probes respectively having different unique base sequences,carrying out PCR on the sample so as to obtain chimeric DNAsrespectively having tag sequence complementary to the detection sequenceof the detection probe having been correlated to the target nucleic acidand the label, hybridizing the chimeric DNAs and the detection probesthrough the detection sequence and the tag sequence, obtaining signalintensity information based on the label on the carrier, and detectingthe target nucleic acid based on the signal intensity information. Themethod for detection according to the disclosure of the of the presentspecification is applied to one or more target nucleic acids and morespecifically, aims to detect the target sequence(s) characteristic inthe target nucleic acid(s). A series of the steps for detection of onetarget nucleic acid is mainly illustrated hereinafter. However, thesteps described below may also be applied for simultaneous detection ofseveral or many target nucleic acids.

(Step of Preparation of Solid Phase Carrier)

The method for detection disclosed in the present specification(hereinafter merely referred to as the present method for detection) maycomprise the step of preparing the solid phase 100 as shown in FIG. 1.The solid phase 100 may be preliminary prepared prior to carrying outthe method for detection, may be commercially obtained or may beprepared every time when carrying out the method for detection.

As shown in FIG. 1, the solid phase 100 may comprise multiple detectionprobes 104 respectively comprising the detection sequences 106 which aredifferent unique base sequences on the carrier 102. Preparation of sucha solid phase 100 may avoid design and synthesis of probes, preparationof arrays and consideration on hybridization conditions.

FIG. 2 shows an example of the solid phase 100. The detection probes 104contain the detection sequences 106 which are respectively unique basesequences for probing. Such detection sequences 106 may be establishedirrespectively of the sequence characteristic to the target nucleic acid10, i.e. the target sequence 12. The detection sequences 106 in thedetection probes 104 are irrespective of the target sequence 12 and maybe established so as to suppress or avoid non-specific binding betweenmultiple detection probes 104 and obtain suitable hybridizationconditions such as temperature and time. In addition, same detectionprobes 104 may be used all the time irrespective of the variation of thetarget nucleic acid 10.

The detection sequence 106 in the detection probe 104 may be basesequences of SEQ ID NO: 1 to SEQ ID NO: 100 or their complementary basesequences. These base sequences have the same base length and have amelting temperature (Tm) of 40° C. or higher and 80° C. or lower, morepreferably 50° C. or higher and 70° C. or lower, thereby givinghomogeneous hybridization results under the same hybridizationconditions.

The detection sequence 106 in the detection probe 104 may beappropriately selected from such candidate base sequences. Two or moredetection probes 104 to be used preferably have melting temperatures asclose as possible to each other. When multiple target nucleic acids 10are exhaustively and simultaneously detected, multiple detection probes104 for respective multiple target nucleic acids 10 are preferablycombined so as to have melting temperatures closest to each other. Forexample, when detection probes 104 are arranged in order of theirmelting temperatures, two or more detection probes 104 for respectivetwo or more target nucleic acids 10 to be distinguished may be selectedfrom two base sequences adjacent in the arrangement by meltingtemperatures. The detection sequence 106 in the detection probe 104 foranother target nucleic acid 10 may be selected from base sequencesimmediately consecutive to or apart from the base sequence which hasalready been selected. It is also preferable to use the base sequenceswhich have consecutive melting temperatures in the arrangement by themelting temperatures for all detection probes for multiple targetnucleic acids 10 to be detected simultaneously.

The melting temperature may be the one calculated according to a GC %method, a Wallace method, a method according to Current Protocols inMolecular Biology (described in Biotechnology Experiments Illustrated 3,Honto ni fueru PCR (Truly amplifiable PCR), Shujunsha, p. 25); however,it is preferably calculated by a Nearest-Neighbor method to whichimpacts of a range of the melting temperature an a concentration of thebase sequence in the present invention may be included. The meltingtemperature by the Nearest-Neighbor method can be easily obtained byusing, for example, software equipped with Visual OMP (Tomy DigitalBiology Co., Ltd.) or software provided by Nihon Gene ResearchLaboratories Inc. (http://www.ngrl.co.jp) (OligoCalculator,http://www.ngrl.co.jp/tool/ngr_tool.html). SEQ ID NO: 1 to SEQ ID NO:100 are arranged in descending order of the melting temperaturescalculated with Visual OMP (0.1 M probe concentration, 50 mM Na⁺ ion and1.5 mM Mg₊ ion).

The detection sequence 106 in the detection probe 104 is called as aorthonormalization sequence and is designed based on the calculations ona consecutive identical length, melting temperature prediction by theNearest-Neighbor method, a Hamming distance, secondary structureprediction on DNA sequences having certain base lengths obtained fromrandom numbers. The orthonormalization sequences are base sequences ofnucleic acids which have homogeneous melting temperatures and thus aredesigned so as to have the melting temperatures in a constant range,which do not inhibit hybridization with the complementary sequencesbecause nucleic acids are structured intramolecularly, and which do notstably hybridize with base sequences other than complementary basesequences. Sequences contained in one orthonormalization sequence grouphardly react or do not react to sequences other than a desiredcombination or within their sequences. When orthonormalization sequencesare amplified by PCR, the amount of the nucleic acids quantitativelyamplified correspond to an initial amount of the nucleic acids havingthe orthonormalization sequences without influenced by a problem such ascross-hybridization as mentioned above. Such orthonormalizationsequences are reviewed in H. Yoshida and A. Suyama, “Solution to 3-SATby breadth first search”, DIMACS Vol. 54, 9-20 (2000) and JapanesePatent Application No. 2003-108126. The orthonormalization on sequencescan be designed by using the methods described in these documents.

The detection probes 104 are fixed on the carrier 102. The carrier 102may be the solid phase carrier. The carrier 102 may be, for example,plastics, glass or any other material without limitation. A shape of thecarrier 102 may be a plate as shown in FIG. 1 or may be a bead withoutlimitation. The solid phase 100 is preferably the array (particularlymicroarray) in which the support 102 is a solid phase plate and multipledetection probes 104 are fixed with a regular sequence. The array can befixed with many detection probes 104 and is suitable for detectingvarious target nucleic acids 10 simultaneously and exhaustively. Thesolid phase 100 may comprise multiple defined array regions on thecarrier 102. On the multiple array regions, the same sets of detectionprobes 104 or different sets of detection probes 104 may be fixed. Whendifferent combinations of the sets of detection probes 104 are fixed onmultiple array regions, individual array regions may be assigned fordetection of target nucleic acids 10 in different genes.

The preferred solid phase 100 may comprise two or more detection probes104 arranged in order of their melting temperatures. For example, byusing such a solid phase 100 in which two or more detection probes 104for two or more target nucleic acids 10 corresponding to two or moretarget sequences 12 which may exist at certain sites in certain genesare arranged in such order, variation in hybridization due to thedifference in melting temperatures of detection sequences 106 indetection probes 104 or to position to where detection probes 104 arefixed is suppressed, thereby allowing accurate detection of targetnucleic acids 10 in the sample.

The detection probes 104 may be fixed by any mode without limitation,which may be covalent or non-covalent. The detection probes 104 may befixed on the surface of the carrier 102 by any various well-knownmethods in the art. The surface of the carrier 102 may compriseappropriate linker sequences. The linker sequences preferably have thesame base length and same sequence for the respective detection probes104.

(Step of Obtaining Chimeric DNA: Step of PCR)

As shown in FIG. 1, the step of PCR may comprise carrying out PCR on thesample so as to obtain the chimeric DNA 60 having the label 42 and thetag sequence 66 which is able to hybridize with the detection sequence106 in the specific detection probe 104 having been correlated to thetarget nucleic acid 10. By obtaining such a chimeric DNA 60, thedetection probe 104 can be employed having the unique detection sequence106 which has been determined in advance regardless of the base sequenceof the target sequence 12 in the target nucleic acid 10. The tagsequence 66 is preferably complementary such that it can specificallyhybridize to the unique detection sequence 106 in the detection probe104, and more preferably completely complementary to the detectionsequence 106. The label is described hereinafter.

Primers used in the step of PCR are not specifically limited as long asthe above chimeric DNA 60 can be obtained. Exemplary preferred step ofPCR in the present method for detection is now described with referringto FIG. 1. The upper right of FIG. 1 shows a step of carrying out PCR onthe target nucleic acid 10 and its complementary strand 20 in the samplewith the first primer 30 and the second primer 40 to obtain anamplification product, chimeric DNA 60.

(First Primer)

As shown in FIG. 1, the first primer 30 contains the identificationsequence 32 and the tag addition sequence 36. The first primer 30 isprepared as many as the target nucleic acids 10. When two kinds ofmutations are expected at a certain part in a genomic DNA of a certainkind of an animal, which are, for example, single nucleotidesubstitutions with A for a wild type and T for a mutation, there are twotarget nucleic acids 10 for this part. Thus, one target nucleic acid 10for this part contains the target sequence 12 having the wild type baseand the other target nucleic acid 10 contains the target sequence 12having the mutated base. Accordingly, when there are two target nucleicacids for a certain site of a gene, two first primers 30 are preparedeach having the identification sequence 32 complementary to the targetsequence 12 in each of the target nucleic acids 10

(Identification Sequence)

The identification sequence 32 can specifically hybridize to the targetsequence 12 which is a characteristic sequence in the target nucleicacid 10, in order to identify the target nucleic acid 10. Theidentification sequence 32 is established to be complementary such thatit can hybridize to the target sequence 12 in the target nucleic acid 10with high selectivity, and preferably is established to be completelycomplementary (specific). The preferred length of the identificationsequence 32 may vary according to mutations and is not specificallylimited, but is preferably 15 bases or more, for example. Theidentification sequence 32 having 15 bases or more in length canhybridize to the target sequence 12 with high selectivity. Theidentification sequence 32 having 60 bases or less in length ispreferable due to reduced non-specific hybridization.

(Tag Addition Sequence)

The first primer 30 may comprise the tag addition sequence 36 for addingthe tag sequence 66 to the amplified product, chimeric DNA 60, so as toallow the chimeric DNA 60 being able to hybridize to the detectionsequence 106 in the detection probe 104. The tag sequence 66 in thechimeric DNA 60 is for detecting the target nucleic acid 10, thus isestablished to be able to hybridize to the detection sequence 106 in thedetection probe 104 for every target nucleic acid 10. Thus, one chimericDNA 60 corresponding to one target nucleic acid 10 is correlated to onedetection probe 104. The tag sequence 66 is preferably completelycomplementary to the unique detection sequence 106 in the detectionprobe 104. Thus, the tag addition sequence 36 preferably has the samebase sequence as the unique detection sequence 106 in the detectionprobe 104 for detection.

As described above, the first primer 30 is prepared so as tospecifically bind to the target sequence 12 in the target nucleic acid10 and is prepared as many as the target nucleic acids 10, therebyspecifically amplifying the target nucleic acids 10 while detecting thesame. The first primer 30 is also formed to allow specific binding ofthe PCR amplified product, chimeric DNA 60, to the particular detectionprobe 104 which has been correlated to the target nucleic acid 10.

(Second Primer)

As shown in FIG. 1, the second primer 40 may contain the label 42 andthe partial sequence 44 which is identical to the base sequence adjacentto the target sequence 12 in the target nucleic acid 10. The label 42may be at the 5′-side of the second primer.

(Label)

The label 42 is for detecting the PCR amplified product chimeric DNA 60.The label 42 may be appropriately selected from well-known labels. Thelabel may be any of various dyes emitting fluorescent signal afterexcitation such as fluorescent substances, or a substance emitting anyof various signal after combining it with a secondary component byenzyme reaction or antigen-antibody reaction. The label may be typicallyfluorescent labeling substances such as Cy3, Alexa 555, Cy5, Alexa 647.The detection by color development may be used by combining biotin andstreptoavidin-HRP and processing them with a substrate.

(Partial Sequence)

The partial sequence 44 has the same base sequence as the partialsequence 14 adjacent to the target sequence 12 in the target nucleicacid 10. The partial sequence 14 adjacent to the target sequence 12 doesnot mean that the partial sequence 14 is immediately at the 5′-side ofthe target sequence 12 without interposing one base (nucleotide)therebetween, but may be the sequence interposing appropriate number ofbases (nucleotides). The partial sequence 44 in the second primer is thesequence allowing annealing of the second primer 40 to the complementarysequence 20 of the target nucleic acid 10.

When a mutation on DNA is detected, the first primer 30 and the secondprimer 40 are designed for the target nucleic acids 10 respectively ofthe wild type and the mutant. In this case, the partial sequence 44 ofthe second primer 40 may be common to these target nucleic acids 10.Namely, the partial sequence 44 may be a common partial sequenceadjacent to the target sequence 12 in these target nucleic acids 10. Thecommon partial sequence is a base sequence which is common regardless ofthe mutation. Due to this, amplification efficiency of the targetnucleic acids 10 can be averaged and the amount of the first primer 40to be used may be dec-eased. The partial sequence 44 may be the sequencehaving homology to multiple target nucleic acids 10 corresponding tomultiple target sequences 12 constituting mutations.

As described above, the second primer 40 contains the label 42 and thepartial sequence 44, and is formed so as to synthesize the chimeric DNA60 containing the target sequence 12 due to the partial sequence 44.When the present method is to detect multiple target nucleic acids 10having multiple target sequences 12 constituting mutations, the secondprimer 40 may have the common partial sequence 44 which allowsamplification of multiple target nucleic acids 10 having multiple targetsequences 12 constituting mutations under the same condition.

The step of obtaining the chimeric DNA 60 with the first primer 30 andthe second primer 40 is now described with referring to FIGS. 1 and 3.In the following description, only PCR reaction which may give thedesired chimeric DNA 60 is explained.

As shown in FIG. 3, the first primer 30 anneals to the target sequence12 in the target nucleic acid 10 through the identification sequence 32.As a result, a new DNA strand is extended from the first primer 30 withthe target nucleic acid 10 as a template, thereby synthesizing a DNAstrand 50 comprising a newly synthesized partial sequence 14 (-). Theobtained DNA strand 50 has the tag addition sequence 36, theidentification sequence 32 and the partial sequence 14 (-).

To the partial sequence 14 (-) in the thus obtained DNA strand 50 thenanneals the second primer 40 through its partial sequence 44. As aresult, a new DNA strand is extended from the second primer 40 with theDNA strand 50 as a template, thereby synthesizing a DNA strand 60comprising a base sequence complementary to the identification sequence32 and a base sequence complementary to the tag addition sequence 36. Asthe identification sequence 32 has identical base sequence as a targetsequence 12 (-), a base sequence complementary to the identificationsequence 32 has the same sequence as the target sequence 12. As the tagaddition sequence 36 is identical to the unique detection sequence 106in the detection probe 104, a base sequence complementary to the tagaddition sequence is the tag sequence 66 which is complementary to thedetection sequence 106 in the detection probe 104. The thus obtained DNAstrand 60 is the chimeric DNA 60 comprising the label 42 and has beencorrelate to the target sequence 12 and the detection probe 104. Thechimeric DNA 60 is used as a template in further amplification reaction.

The step of PCR for obtaining the chimeric DNA 60 is preferably the stepof asymmetric PCR. Asymmetric PCR can be carried out by varying theconcentrations of the first and second primers, for example.

As the chimeric DNA 60 is obtained as a double-stranded DNA, it isdissociated to single strands for subjecting them to the step ofhybridization. The dissociation in this context can be achieved by adenaturing treatment comprising chemical denaturation and thermaldenaturation. When oligonucleotides linked are dissociated by chemicaldenaturation, a treatment known to a person skilled in the art such asalkaline denaturation may be carried out. When oligonucleotides linkedare dissociated by thermal denaturation, they may be placed under atemperature of 85° C. or more, preferably 90° C. or more underphysiological conditions; however, a person skilled in the art canselect appropriate dissociation method.

According to the step of PCR in which the sample which may possiblycontain the target nucleic acid 10 is subjected to the step of PCR,chimeric DNAs 60 can be obtained at once which can specifically detectthe target nucleic acids 10 via the detection probes 104 having beencorrelated to the target nucleic acid 10.

A PCR reaction product may be subjected to a next step withoutcollecting chimeric DNAs 60, because only chimeric DNAs 60 can bind tothe detection probes 104 which are then detected through the label 42.Chimeric DNAs 60 may be collected by a well-known method. For example,the chimeric DNAs 50 may be separated and collected by a well-knownmethod such as using an appropriate solid phase carrier after beingdissociated into single strands.

(Step of Hybridization)

The step of hybridization is the step in which the detection probes 104having the detection sequences 106 complementary to the tag sequences 66in the chimeric DNAs 60 on the solid phase 100 fixed on the carrier 102and the chimeric DNAs 60 are brought into contact so as to allowhybridization. As shown in FIGS. 1 and 2 (c), when the chimeric DNA 60is complementary to the detection sequence 106 in the detection probe104 such that they can specifically hybridize each other under certainconditions, they hybridize each other to form a double-strand at acertain detection probe 104 on the carrier 102 in this step. A washingstep may further be appropriately contained following to the step ofhybridization.

To the step of hybridization is provided the chimeric DNA 60 which hasbeen synthesized in the step of PCR only when the target nucleic acid 10is present in the sample and which hybridizes only to the detectionprobe 104 having been correlated. The detection sequence 106 in thedetection probe 104 and the tag sequence 66 in the chimeric DNA 60 areselected with high selectivity so that mishybridization is highlysuppressed, thereby highly suppressing non-specific hybridization of thechimeric DNA 60 to the detection probe 104 in the step of hybridization.

(Step of Obtaining Signal Intensity Information)

The step of obtaining signal intensity information is the step in whichsignal intensity information about the target nucleic acid 10 hosed inthe label 42 on the carrier 102 is obtained after hybridization.According to the present step of obtaining signal intensity information,the chimeric DNA 60 hybridizes to the detection probe 104 to providesignal intensity information based on the label 42.

As shown in FIG. 1, in the step of obtaining signal intensityinformation, signal 48 derived from the label 42 associated with thedetection probe 104 on the solid phase 100 may be detected. As theposition of the detection probe 104 correlated has been already known onthe solid phase 100, the presence or absence or ratio of the targetnucleic acid 10 can be determined by detecting the signal 48 in the neststep of detection.

The step of obtaining signal intensity information may be carried out byselecting a conventional well-known method according to the form of thecarrier 102 or the label 42. Typically, after removing non-hybridizedoligonucleotides and the like from the carrier 102 by washing,fluorescent signal of the added labeling substance may be detected withan array scanner and the like or the labeling substance may be subjectedto chemical luminescence reaction. When the carrier is a bead, adetection method using a flow cytometer may be employed.

(Step of Detection)

The step of detection is the step in which the presence or absence orratio of the target nucleic acid 10 in the sample is detected based onsignal intensity information of the label 42 obtained for the detectionprobe 104. According to the present method, even when multiple targetnucleic acids 10 are detected simultaneously, the target sequences canbe surely detected. According to the present method, as non-specificbinding to the detection probe 104 is highly suppressed in the step ofhybridization, the target nucleic acid 10 can be accurately detectedwith high detection sensitivity and the presence or absence or ratiothereof can be obtained.

(Primer Set)

The primer set of the present invention comprises the first and secondprimers described hereinabove. The primer set is used in combinationwith the solid phase on which the detection probes 104 have been fixed,and is suitable for obtaining the chimeric DNA described hereinabove.The first primer comprises the identification sequence 32 which isspecific to the particular target nucleic acid for detecting a mutationamong individuals regarding the same gene and the like or a differencebetween species or genera and the tag addition sequence 36 having beencorrelated to the detection sequence 106. The second primer comprisesthe label. The primer set may be for detecting two or more targetnucleic acids. In this case, the primer set may comprise the firstprimers specific to respective target nucleic acids and the singlesecond primer common to two or more target nucleic acids. The primer setof the present invention may be comprised in a kit together with thecarrier such as the array to which the detection probes describedhereinabove have been fixed.

EXAMPLE 1

The present invention is specifically described with the followingexamples, which do not limit the present invention.

EXAMPLE 2

The target nucleic acid was detected with the method for detection ofthe present invention in the present example according to the followingprocedures, which are now described step by step.

-   (1) Preparation of DNA microarray-   (2) Preparation and amplification of target nucleic acids and    primers-   (3) Hybridization-   (4) Detection with scanner-   (5) Data analysis

(1) Preparation of DNA Microarray

On a plastic plate, aqueous solutions of synthetic oligo DNAs (NihonGene Research Laboratories Inc.) modified at a 3′-end with an aminogroup were spotted as the detection probes using a GENESHOT® spotter atNGK Insulators, Ltd. As shown in Table 1, 100 synthetic oligo DNAs wereused which were D1_(—)001 to D1_(—)100 shown in Supplementary Table 1 ina document (Analytical Biochemistry 364 (2007) 78-85) (see FIG. 5).After spotting, the plate was baked at 80° C. for an hour. These probesare arranged in descending order of melting temperatures correspondingto Tm calculated with Visual OMP (0.1 M probe concentration, 50 mM Na+ion and 15 mM Mg+ ion).

The synthetic oligo DNAs were fixed according to the followingprocedures. Namely, the plate was washed with 2×SSC/0.2% SDS for 15minutes, with 2×SSC/0.2% SDS at 95° C. for 5 minutes, before three timesof washing with sterilized water (mixing by turning vertically for 10times). The plate was then dried by centrifugation (1000 rpm×3 minutes).

(2) Preparation of Target Nucleic Acids and Primers and Amplification

Sample genes to be detected were derived from two types of oralmicroorganisms, Enterococcus faecalis (sample 1) and Pseudorambibacteralactolyticus (sample 2). The length of these samples was about 150 bp,which surrounded characteristic sequences of microorganism, andartificial genes having these base sequences were used as target nucleicacids. Primers for amplifying these target nucleic acids wereartificially synthesized as follows. The second primer, i.e. the forwardprimer (F primer) was 5′-AGGTTAAAACTCAAAGGAATTGACG-3′ (SEQ ID NO: 101),which was labeled with Cy3 at the 5′-side. The first primers, i.e. thereverse primers (R primers) were prepared according to the targetsequences of the samples. The reverse primer for the sample 1 was5′-GCAGATTCATTGGTCAGAGAACATATCTCTAGAGTGGT-3′ (SEQ ID NO: 102) and thereverse primer for the sample 2 was5′-CATCTAAAGCGTTCCCAGTTCCATATCTCTATTGCGCT3′ (SEQ ID) NO: 103).

These samples were amplified as follows. A reagent used for amplifyingthe samples was a multiple PCR kit from QIAGEN. A thermal cycler usedwas GeneAmp PCR System 9700 from Applied Biosystems.

The following reagents were prepared for each sample. The F primer and Rprimers used were respectively adjusted to 10 μmol/μl.

-   (Reagent Preparation)-   dH₂O 15.0 μl-   multiple PCR kit 25.0 μl-   F primer 3.75 μl-   R primer 3.75 μl-   Sample 2.5 μl-   Total 50.0 μl

The prepared reagents were transferred to a thermal cycle plate andthermal cycle reaction (95° C. for 15 min; then 50 cycles of 94° C. for30 see, 62° C. for 30 see and 72° C. for 30 min 72° C. for 10 min, anddecreased to 4° C.) was carried out. The amplified labeled samples werepurified with MinElute PCR Purification Kit from QIAGEN, beforeverifying that amplified products had a desired length.

(3) Hybridization

In order to hybridize the amplified samples obtained in (2) with thedetection probes fixed on the microarray, the following Hybri controland Hybri solution were prepared, which were used for preparation of ahybridization reagent. An Alexa 555-labeled oligo DNA sequence used forHybri control was Alexa555-rD1_(—)100 which was obtained by labeling the5′-end of a complementary sequence of D1_(—)100, among those probesdescribed in FIG. 5, with Alexa 555.

(Hybri Control)

-   Alexa555-rD1_(—)100 10 μl-   TE (pH 8.0) 390 μl-   Total 400 μl

(Hybri Solution)

-   20×SSC 2.0 ml-   10% SDS 0.8 ml-   100% Formamide 12.0 ml-   100 mM EDTA 0.8 ml-   milliQ 24.4 ml-   Total 40.0 ml

(Reagent for Hybridization)

-   Hybri control 1.5 μl-   Hybri solution 9.0 μl-   Subtotal 10.5 μl-   Labeled sample 7.5 μl-   Total 18.0 μl

A prepared labeled sample solution was heated in GeneAmp PCR system 9700from Applied Biosystems at 90° C. for 1 minute prior to heating in aheat block (TAITEC, DTU-N) at 80° C. for 1 minute. The sample solutions(9 μl each) were deposited on a spotted area of the microarray and leftto stand at 37° C. for 30 minutes for hybridization reaction whilepreventing evaporation with Thermoblock Slide for Comfort/plus(Eppendorf).

(Washing)

After hybridization, the microarray substrate after hybridizationreaction was soaked in a glass staining vat filled with washing solutionhaving the following composition, incubated with vertical shaking for 5minutes, and the glass substrate was transferred to a glass staining vatfilled with sterilized water, incubated with vertical shaking for 1minute, and dried by centrifugation at 2000 rpm for 1 minute to removeremaining water on the surface of the microarray substrate.

-   (Composition of Washing Solution)-   milliQ 188.0 ml-   20×SSC 10.0 ml-   10% SDS 2.0 ml-   Total 200.0 ml

(4) Detection with Scanner

Fluorescent images were obtained with ArrayWoRx from Applied Precision,Inc. by appropriately adjusting time of exposure. Fluorescent signalfrom the obtained images were converted to numerical values with GenePixPro.

(5) Data Analysis

Fluorescent signal from the obtained images were converted to numericalvalues with GenePix Pro, which is software for numerical conversion ofimages. FIG. 6 shows the results of verification on whether or not thesamples 1 and 2 respectively bound non-specifically to the detectionprobes.

As shown in FIG. 6( a), the reaction with the mixture of the samples 1and 2 gave fluorescent signal for both probes, indicating that thesamples were detected. As shown in FIGS. 6( b) and 6(c) in which eitherof the sample 1 or sample 2 was subjected to the reaction withoutmixing, it was found that non-specific binding to an undesired probe wassignificantly decreased. It was also found that each sample specificallybound to the respective detection probes designed to identify therespective samples.

Next, a conventional detection method of target nucleic acids (methoddescribed in Non-patent document 4) was verified as a comparativeexample. In the following comparative example, the target nucleic acidwas detected with the conventional detection method according to thefollowing procedures, which are now described step by step.

On a plastic plate, aqueous solutions of synthetic oligo DNAs (NihonGene Research Laboratories Inc.) modified at the 3′-end with an aminogroup were spotted as the detection probes using a GENESHOT® spotter atNGK Insulators, Ltd. The used synthetic oligo DNA sequences were5′-ACCACTCTAGAGATA-3′ (SEQ ID NO: 104) for a sample 1, and5′-AGCGCAATAGAGATA-3′ (SEQ ID NO: 105) for a sample 2. After spotting,the plate was baked at 80° C. for an hour, and the DNAs were arranged indescending order of Tm.

Sample genes to be detected were the same samples 1 and 2 used in theexample. Common primers for amplifying the target nucleic acids wereartificially synthesized as follows. The F primer was5′-AGGTTAAAACTCAAAGGAATTGACG-3′ (SEQ ID NO: 106), which was labeled withCy3 at the 5′-side. The R primer was 5′-ATGGTGTGACGGGCGGTGTGT-3′ (SEQ IDNO: 107).

The samples 1 and 2 were amplified by the thermal cycle reaction asdescribed in the above (3) to (5), and hybridized with the detectionprobes prepared in Example 6 before washing and signal detection. FIG. 7shows the results of verification on whether or not the samples 1 and 2respectively bound non-specifically to the detection probes.

As shown in FIGS. 7( a) to 7(c), weak signal was detected with thedetection probe for the sample 1 even when the sample did not containthe sample 1, and weak signal was detected against the detection probefor the sample 2 even when the sample did not contain the sample 2,which were thus showing non-specific binding of the samples.

The time required for hybridization in the conventional method was abouttwo hours. Non-specific reaction to the detection probes was observed(about 10% in fluorescent intensity). On the other hand, the timerequired for hybridization in the present invention was decreased toabout 30 minutes and non-specific reaction of the samples to undesireddetection probes on the DNA microarray could be significantly reduced(less than 1% in fluorescent intensity). Thus, according to the presentinvention, hybridization can always be carried out at a constanttemperature (about 37° C.) in about 30 minutes of tune (one-fourth ofthe conventional method). The present invention can also provide resultswith more intense signal than the conventional method, and allows moreaccurate detection of bases in a particular nucleic acid and moreaccurate determination of sequence than the conventional method. Theconventional method sometimes requires optimization of hybridizationconditions, re-design of probe sequences or re-preparation of arraysuntil desired result are obtained. On the other hand, the presentinvention does not require re-design of probe sequences or re-productionof arrays and allows examination with arrays having the samespecification all the time.

[Sequence Listing Free Text]

SEQ ID NOs: 1 to 100: probes, SEQ ID NOs: 101 to 103: primers, SEQ IDNOs: 104 and 105: probes, SEQ ID NOs: 106 and 107: primers

[Sequence Listing]

The invention claimed is:
 1. A method for detection of a target nucleicacid in a sample, comprising: preparing a solid phase comprisingdetection probes respectively having different base sequences; carryingout PCR on the sample to obtain chimeric DNAs each having a label, and atag sequence complementary to each of the detection probes having beencorrelated to the target nucleic acid; bringing the chimeric DNAs intocontact with the detection probes such that the chimeric DNAs and thedetection probes can hybridize through the tag sequences; obtainingsignal intensity information based on the label on the solid phase; anddetecting the target nucleic acid based on the signal intensityinformation; wherein the step of PCR comprises: preparing a set ofprimers which does not include a universal primer, wherein the set ofprimers consists of a first primer having an identification sequencecomplementary to a target sequence in the target nucleic acid and a tagaddition sequence complementary to the tag sequence, and a second primerhaving a partial sequence that has the same sequence as a partialsequence adjacent to the target sequence and the label; and carrying outPCR on the sample using a set consisting of the first primer and thesecond primer to synthesize the chimeric DNA having the target sequence,the tag sequence and the label.
 2. The method according to claim 1,wherein the step of PCR is the step of using, for two or more targetnucleic acids, two or more first primers and one second primer common tothe two or more target nucleic acids.
 3. The method according to claim1, wherein the step of PCR is the step of amplifying the chimeric DNAsby asymmetric PCR.
 4. The method according to claim 1, wherein thetarget nucleic acids have a target sequence other than SNPs.